The present invention concerns methods and devices for observing or analysing samples on a support. More particularly, it concerns a device for sequential observation of several samples arranged side by side on a common support, notably a common plate.
Such device, notably of the microscope type, comprises an objective for observing at least part of a sample along an observation axis from an observation surface of the support and a stage for positioning the support adapted to ensure a relative displacement between the support and the observation axis in a plane perpendicular to the observation axis but leaving free the displacement along the presumed vertical observation axis. It further comprises means for illuminating at least part of a sample and means for acquiring an image at the objective output.
Such a device is used notably for rapidly analysing a large number of samples arranged on a common support, such as a plate, such samples being constituted, for example, of cells, in particular adherent, mammalian, prokaryotic, plant cells, etc. or other organisms, for instance pathogens (viruses, etc).
Rapid cellular analysis on a large number of samples is a necessity in the pharmaceutical industry, for high throughput screening of new active substances, and in the cosmetics industry, where cellular models are used to test new substances and control manufactures. These industries would like to make greater use of cellular tests and are asking that the implementation thereof be simplified.
The culture plates commonly used to hold biological (notably cellular) samples are generally tray-like in shape. They have a series of adjacent cells, called wells, in all or part of which is contained a sample. These wells are generally arranged in parallel axes to each other and extend along the thickness of the plate. They open at the top surface of the plate and are sealed by a bottom which generally forms the bottom surface of the plate. The bottoms of adjacent wells are therefore generally joined to one another. Thus, the bottom surface of the plate is essentially continuous. Other supports for the analysis of biological samples are for example membranes, glass slides, filters, or any other translucid or opaque support, etc.
To observe samples under a microscope, the plate (or any other support) is placed above or below the objective of the microscope whose axis is oriented vertically. Observation occurs through the bottom of each well, i.e. through the bottom surface of the plate, such bottom surface constituting the observation surface, or directly above the sample. The samples are brought one by one before the objective by displacing the plate in a plane perpendicular to the observation axis by means of a positioning stage. The latter is adapted to hold the plate primarily or solely at its periphery, thus leaving free the observation surface of the plate. In addition, the stage is adapted to leave the plate a certain range of motion in the observation axis.
Plates are generally made of poured or moulded material, usually polystyrene. For this reason, plates more often than not comprise defects in the flatness of their bottom surface forming the bottom of the wells. The size of such defects largely exceeds the depth of field of the objective. The difference between extreme points on the bottom surface commonly reaches 0.2 to 0.3 mm.
The defects in the flatness of the bottom surface of the plate and its deformation cause variations in the distance between the objective and the sample to be analysed. It is therefore necessary, in order to observe an entire plate or samples arranged in wells covering a large section of the plate surface, to frequently adjust the focus of the objective to compensate the variation in this distance.
This drawback is seen for example in standard fluorescence microscopes, equipped with a CCD camera and morphometric software. The same problem arises for phase contrast examination such as proposed by certain instruments.
Automatic focusing on this type of instrument is slow and unreliable: an empty well can cause instability of the system or even erroneous focusing on a fault in the external surface.
The need to frequently adjust the focus of the objective lowers the speed of image acquisition of the samples.
An analogous problem arises during the analysis of microparticles which are increasingly used as effectors of biochemical reactions, when such microparticles are deposited in the wells of a plate.
Likewise, the search for bacteria on filters requires frequent adjustment of the focus of the objective due to defects in the flatness of the filter.
Another drawback is the use of mercury and/or halogen lamps, which do not afford stable and uniform illumination.